This project's long-term objective is to develop microscopy tools that can advance our understanding of intracellular transport, particularly as it relates to neurological diseases such as Alzheimer's and Amyotrophic lateral sclerosis (ALS). Our specific aims are: 1) to combine our newly developed, motion-enhanced differential interference contrast (MEDIC) microscopy with simultaneous fluorescence imaging;2) to calibrate a novel method that uses MEDIC images to determine the sizes of subdiffraction-limit intracellular particles;and 3) to apply these new tools to obtain in vivo force-velocity curves for known vesicle-transporting molecular motors. Successful completion of these aims will provide new microscopic tools and deeper insight into how living cells use load-sharing between cooperating motors to increase transport velocity to regulate important functions, such as synaptogenesis, chemotaxis, mitosis, and apoptosis. To achieve Aim 1, we will extend our preliminary results, which used a commercial image splitter to provide simultaneous acquisition of MEDIC and epi- fluorescence signals (epi-MEDIC), to develop a combined MEDIC-TIRF (total internal reflection fluorescence) system as well as a short-wave DIC system that uses blue light instead of near-infrared for the MEDIC channel. Aim 2 will build on our preliminary calibration curve of 0.1-1.2 micron-diameter polystyrene beads and validate our sizing algorithms on spherical vesicles. Aim 3 will use MEDIC-TIRF and epi-MEDIC to image NT2 cells that have fluorescently labeled vesicular organelles (e.g., lysosomes) and to obtain a plot of vesicle diameter vs. velocity, which we will convert into a force-velocity curve via Stokes'Law an generalized Stokes-Einstein calculations of intracellular viscosity. PUBLIC HEALTH RELEVANCE: improve understanding of the subcellular dynamics that underlie healthy and pathological states, our novel light microscopy tools will enable faster live-cell imaging of labeled and unlabeled structures. They will enhance the contrast of moving intracellular particles and facilitate their molecular characterization, which will especially aid efforts to understand the biophysical properties of molecular motors and the role they play in neurodegenerative diseases. R15 APPROPRIATENESS: For each of the past 5 years, 3-12 undergraduate and 1-2 graduate students have conducted research and gained state-of-the-art training in our laboratory. Project aims are modular and, based on the successful preliminary results obtained by students in our laboratory (and published as co-authors in peer reviewed journals), will be ideal for significant student involvement in meritorious research.